.alpha.1 Antitrypsin (AAT) is a 52 kDa plasma serine protease inhibitor. Its normal plasma concentration ranges from 150 to 350 mg/dl (Brantly et al., 1988), although it behaves as an acute phase reactant increasing 3-4-fold during host response to inflammation and/or tissue injury such as with pregnancy, acute infection, tumors, estrogen, and typhoid vaccine (Kushner, 1988; Schreiber, 1987). AAT is capable of inhibiting a variety of proteases including trypsin, chymotrypsin, plasmin, thrombin, kallikrein, factor Xa, plasmogen and cathepsin G (Carrel et al., 1986; Laurell & Jeppson 1975; Travis & Salvesen, 1983), but its main physiological role is the inhibition of neutrophil elastase. Neutrophil elastase is not only capable of attacking elastin but may also cleave other connective tissue proteins, such as type I, III, and IV collagens, the protein portion of proteoglycans, and laminin (Bieth, 1986). AAT, however, prevents such degradation by forming a tightly bound 1:1 enzyme:inhibitor complex resulting in the slow proteolytic cleavage of the reactive center of the inhibitor between MET-358 and SER-359 of AAT (See SEQ ID NO:1).
The reactive center of human AAT (See SEQ ID NO:1) is contained within an exposed peptide from (ALA-350) to (SER-359) in a stressed loop configuration (Carrell, 1986; Bruch et al., 1988), which may be comparable to the bait region of .alpha.-2-macroglobulin (S-Jensen, 1987). Every serine proteinase so far examined, including bacterial, plant, and reptilian as well as mammalian enzymes, has been reported to disrupt bonds within this loop of the native inhibitor. Cathepsin L (Johnson et al., 1986) and Serratia marcescens metalloproteinase (Virca et al., 1982), for example, have been reported to cleave the peptide bond between MET-358 and SER-359, while Pseudomonas aeruginosa (Morihara et al., 1984), macrophage elastase (Banda et al., 1985) and PNM collagenase (Knauper et al., 1990) have been reported to cleave one amino acid residue N-terminal at the peptide bond between PRO-357 and MET-358. Cathepsin L (Johnson et al., 1986) and Staphylococcus aureus cysteine and serine proteinase (Potempa et al., 1986) have also been reported to cleave the peptide bond between GLU-354 and ALA-355, while Staphylococcus aureus metalloproteinase (Potempa et al., 1986), secreted PMN metalloproteinase (Desochers and Weiss, 1988, Vissers et al., 1988), and PMN collagenase (Knauper et al., 1990) have been reported to cleave two amino acids N-terminal at the peptide bond between PHE-352 and LEU-353. In addition, Crotialus adamenteus (The Eastern Diamondback Rattlesnake) venom proteinase II has been reported to cleave the peptide bond between ALA-350 and MET-351. While cleavage of the inhibitor does not effect the inhibition of the bound AAT, the consensus is that such fluid-phase cleavage inactivates the inhibitor from inhibiting other proteases. (Michaelis et al., 1990; Knauper et al., 1990). There currently is no known function of the smaller cleaved peptide, although it may bind hepatoma and monocyte receptors (Perlmutter et al, 1990).
Kress et al., 1989, report that venom proteinase II from the Eastern Diamondback Rattlesnake cleaves AAT in-vitro between ALA-350 and MET-351 (See SEQ ID NO:1). Additionally, Kress et al., disclosed that one of the fragments produced in the cleavage has the NH.sub.2 terminal sequence: Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn (SEQ ID NO:3). Kress et al., disclose no activity for the fragment and disclose that the fragment does not inhibit the cleavage of trypsin by an elastase.
This invention provides the discovery that a 44-residue, C-terminal fragment of AAT (SEQ ID NO:1) (hereinafter designated "SPAAT" (SEQ ID NO:4)) exists in human subjects. This fragment appears to represent the same fragment disclosed by Kress et al. when AAT was cleaved by snake venom proteinase II. However, despite the absence of activity being attributed to any small fragment of cleaved AAT in the literature and the finding in Kress et al. of no activity associated with the fragment, this invention provides the surprising discovery that SPAAT (SEQ ID NO:4) is in fact a potent inhibitor of elastase. Additionally, the invention provides the completely unexpected discovery that SPAAT (SEQ ID NO:4), when bound by an extracellular matrix protein such as a collagen, inhibits elastase to a much greater degree than SPAAT (SEQ ID NO:4) alone.
Since SPAAT (SEQ ID NO:4), or equivalent polypeptides can be synthesized, the invention provides a much needed effective and inexpensive method to treat conditions such as emphysema and respiratory distress syndrome. Additionally, because of the discovery that the unique association of SPAAT (SEQ ID NO:4) with the extradellular matrix (ECM) greatly increases the half-life, SPAAT (SEQ ID NO:4) can be administered much less frequently than AAT (SEQ ID NO:1). This leads to less expensive administration and greater quality of life for the patient.
Finally, since the invention demonstrates that in vivo SPAAT (SEQ ID NO:4) is bound or deposited on biologically susceptible ECM proteins, such as elastin or collagen, SPAAT (SEQ ID NO:4) can be used in protecting these proteins from the inappropriate attack of enzymes like HNE.